Adenylate Energy Charge Research Articles

5-Hydroxydecanoate Abolishes Cardioprotective Effects of a Structural Analogue of Apelin-12 in Ischemia/Reperfusion Injury

Chemically modified peptide apelin-12 (MA) with enhanced resistance to degradation by proteolytic enzymes is able to protect the heart against myocardial ischemia and reperfusion. This study was aimed to explore the role of mitochondrial ATP-sensitive K+-channels (mitoKATP) in effects of MA on myocardial energy state and membrane integrity in ischemia/reperfusion (I/R) injury. Isolated perfused working rat hearts were used to simulate global ischemia and reperfusion. Acute myocardial infarction was induced by coronary artery occlusion followed by restoration of coronary blood flow in anesthetized rats. Myocardial infarct size and cardiac dysfunction were used as indices of I/R injury at the end of reperfusion. Co-infusion of 5-hydroxydecanoate (5HD), the mitoKATP blocker, along with MA before ischemia significantly decreased functional recovery of isolated hearts as compared to administration of MA alone. These effects were accompanied by increased LDH release in the myocardial effluent, reduced restoration of myocardial ATP, AN, Cr, adenylate energy charge (AEC), and lactate accumulation. Coadministration of 5HD and MA at the onset of reperfusion substantially reduced infarct-limiting effect of the peptide in rats in vivo and increased the plasma LDH and CK-MB activity compared with MA treatment. Additionally, 5HD abolished MA influence on the metabolic state of the area at risk (AAR) at the end of reperfusion. In this case, the contents of metabolites and AEC in the AAR did not differ significantly from the values in control. Therefore, restoration of myocardial energy metabolism and sarcolemma integrity via activation of mitoKATP may be of critical importance for MA-induced protection against I/R injury.

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.

ABSTRACTMammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa.

N-acetylcysteine improves the growth performance and intestinal function in the heat-stressed broilers

This study was carried out to investigate the effects of N-acetylcysteine (NAC) on growth performance and intestinal function of heat-stressed broilers. A total of two hundred 1-d-old Cobb male chicks were allocated to 1 of 4 treatments, with 5 replicated pens per treatment and 10 birds per pen. The experiment consisted of 4 treatments in a 2×2 factorial arrangements with two diets (basal diet or 1g/kg NAC diet) and two temperatures (thermoneutral or heat stress). From day 8–35 of age, broilers were raised at thermoneutral (26±1°C) or exposed to cyclic heat stress (36±1°C from 0800 to 1800 and 26±1°C from 1800 to 0800). The results showed that heat stress reduced ADFI, ADG, plasma concentrations of triiodothyronine (T3) and thyroxine (T4), intestinal villus height (VH), ratio of VH to crypt depth (CD), intestinal mucosal ATP level, adenylate energy charge (AEC), activities of alkaline phosphatase (AKP), antioxidative and digestive enzymes, and mRNA level for Bcl-2, whereas increased the feed/gain, mortality rate, plasma corticosterone level, intestinal CD, intestinal mucosal AMP and malondialdehyde (MDA) levels, and mRNA levels of heat shock protein (HSP70), caspase-3, AMP-activated protein kinase (AMPK), heme-oxigenase (HMOX), and xanthine oxidoreductase (XOR). Dietary supplementation with NAC decreased the feed/gain, mortality rate, plasma corticosterone level, MDA concentration and intestinal mucosal mRNA levels of HSP70, AMPK and HMOX, while elevated the ratio of VH to CD, ATP, catalase (CAT) and trypsine activity in the small intestine of heat-stressed broilers. Taken together, these results suggest that dietary supplementation of 1g/kg NAC could improve growth performance and intestinal function of broilers exposed to heat stress.

The bactericidal effect of an ionizer under low concentration of ozone.

BackgroundSeveral mechanisms have been suggested for the bactericidal action of ionizers including electrical phenomena, effects of negative and positive ions and electrostatic repulsion. Negative and positive ions have indeed been shown to have bactericidal effects. In addition, since ozone is generated along with ions, these may contribute to the bacterial killing. In this study, we used a newly developed ionizer, which generates a relatively low concentration of ozone, to determine whether its effect on bacterial cells were due to ions or ozone, and, if ions, how the ions exerted their effects.ResultsThe effect of ions on bacterial killing was compared with that of the ozone produced using an ion trap to remove the ions. The ionizer had the ability to kill the bacteria, and ion capture dramatically reduced its bactericidal effect, indicating that the ozone generated had little or no bactericidal effect under these conditions, and the ions produced were responsible for almost all the bacterial killing. Operation of the ionizer increased the level of 8-oxo-dG, a marker of oxidative DNA damage, and decreased aconitase activity, which is known to be sensitive to ROS. The ionizer further affected the adenylate energy charge of bacterial cells. Removal of the ions with the ion trap greatly reduced all these effects.ConclusionThese results indicate that negative and positive ions generated by the ionizer are responsible for inducing oxidative stress and so reducing bacterial survival.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0785-5) contains supplementary material, which is available to authorized users.

Nutrient removal by Chlorella vulgaris F1068 under cetyltrimethyl ammonium bromide induced hormesis.

Toxicants are generally harmful to biotechnology in wastewater treatment. However, trace toxicant can induce microbial hormesis, but to date, it is still unknown how this phenomenon affects nutrient removal during municipal wastewater treatment process. Therefore, this study focused on the effects of hormesis induced by cetyltrimethyl ammonium bromide (CTAB), a representative quaternary ammonium cationic surfactant, on nutrient removal by Chlorella vulgaris F1068. Results showed that when the concentration of CTAB was less than 10ng/L, the cellular components chlorophyll a, proteins, polysaccharides, and total lipids increased by 10.11, 58.17, 38.78, and 11.87%, respectively, and some enzymes in nutrient metabolism of algal cells, such as glutamine synthetase (GS), acid phosphatase (ACP), H(+)-ATPase, and esterase, were also enhanced. As a result, the removal efficiencies of ammonia nitrogen (NH4 (+)) and total phosphorus (TP) increased by 14.66 and 8.51%, respectively, compared to the control during a 7-day test period. The underlying mechanism was mainly due to an enhanced photosynthetic activity of C. vulgaris F1068 indicated by the increase in chlorophyll fluorescence parameters (the value of Fv/Fm, ΦII, Fv/Fo, and rETR increased by 12.99, 7.56, 25.59, and 8.11%, respectively) and adenylate energy charge (AEC) (from 0.68 to 0.72). These results suggest that hormesis induced by trace toxicants could enhance the nutrient removal, which would be further considered in the design of municipal wastewater treatment processes. Graphical abstract The schematic mechanism of C. vulgaris F1068 under CTAB induced hormesis. Green arrows ( ) represent the increase and the red arrow ( ) represents the decrease.

Responses to thermal and salinity stress in wild and farmed Pacific oysters Crassostrea gigas

The Pacific oyster Crassostrea gigas was introduced from Japan to many countries in the world for oyster farming, resulting in the establishment of wild populations in intertidal zones and resource competition with local faunas. This study examined physiological responses of wild oysters and farmed oysters to thermal (15°C, 25°C, 37°C and 44°C) and salinity stress (39, 50 and 60ppt). The wild oysters produced more 72kDa heat shock proteins when the temperature increased from 15°C to 25°C and 37°C and the salinity increased from 39 to 50 and 60ppt. However, the amount of 69kDa heat shock protein was similar between farmed and wild oysters when the temperature increased from 15°C to the sublethal temperature 37°C, but it was lower in wild oysters than in farmed oysters when the temperature increased from 15°C to the lethal temperature 44°C. In the tissues, wild oysters used more glycogen to promote metabolic activities by increasing the level of AEC (adenylate energy charge). The results suggest that farmed oysters might have limited ability to cope with heat stress due to low energy reserve and glycolysis activity for HSP synthesis. This study provides experimental evidence on differential responses between wild and farmed oysters to temperature and salinity changes, leading to a better understanding on the pattern of distribution for invading oyster species in the marine environment and the adaptation of marine invertebrates to the threat of climate change.

Hypothermal effects on survival, energy homeostasis and expression of energy-related genes of swimming crabs Portunus trituberculatus during air exposure

Previously, dry or semi-dry approach under the hypothermal condition is proved to be an alternative method in transport of live swimming crabs Portunus trituberculatus. However, we wondered whether this method can improve crab survival when temperature is kept as cool as possible. In this study, we hypothesized that there is a thermal threshold below which dry or semi-dry approach (air exposure) could cause crab physiological disruption and therefore aggravate their mortality. To test the above hypothesis, crabs (23°C) were exposed to air at temperatures ranging from 4 to 16°C. Results showed that crabs had a worse survival and vigor at temperatures below 12°C. Then we tested crab energy metabolism to explore the possible reason. It was shown that total adenine nucleotide and adenylate energy charge in gills were remarkably reduced by air exposure of below 12°C. This increased the need for crabs to re-balance energy metabolism, which was indicated by the upregulation of AMPKα and HIF-1α. Meanwhile, there was a significant increase of the expression of Na+/K+-ATPase, V-type ATPase and HSP90 at temperatures below 12°C, while all treatments shared a similar level of hemocyanin, urate and lactate in hemolymph and expression of cytochrome c oxidase and NADH-ubiquinone reductase in gills. These results implied that dry or semi-dry approach below 12°C could exert detrimental effects on P. trituberculatus, and perturbation of energy homeostasis, which is more related with changes of energy-demanding physiological pathways, is a possible reason of crab death and poor vigor.

Effects of stunning/slaughtering methods in rainbow trout (Oncorhynchus mykiss) from death until rigor mortis resolution

The effects of stunning/slaughtering methods (carbon monoxide asphyxia, CO; electroshock, E; asphyxia in the air, A) were studied in rainbow trout kept in freshwater at 12°C. The fish were analysed for blood parameters, rigor mortis evolution, fillets shape changes over 48h, and ATP depletion and Adenylate Energy Charge (AEC) in muscle immediately after death. Treatment A was most stressful giving the lowest pH values during rigor evolution, the highest concentration of lactate (6.31mM) in plasma and the lowest AEC values in the muscle.None of the three-stunning/slaughtering methods affected perimeter, length and height of the fillets, with exception for the area at 24h and 48h post mortem, which was significantly reduced by CO treatment (CO<A<E). Globally, CO and E treatments showed the highest ability in preserving muscle energy immediately after death, whereas treatment E resulted the most suitable method for slaughtering, based on blood and muscle indicators. Statements of relevance1.The application of CO as slaughtering/killing method on rainbow trout, focusing on the effects produced in the pattern of some analytical parameters during the first period post mortem2.The use of carbon monoxide for killing fish has been only recently experimented and only in Atlantic salmon.3.Improve the knowledge about the use of CO, due the scarcity of information in scientific literature

Juvenile roach (Rutilus rutilus) increase their anaerobic metabolism in response to copper exposure in laboratory conditions.

This study aims to determine the potential impairment of cell energy synthesis processes (glycolysis and respiratory chain pathways) by copper in juvenile roach at different regulation levels by using a multi-marker approach. Juvenile roach were exposed to 0, 10, 50, and 100µg/L of copper for 7days in laboratory conditions. The glycolysis pathway was assessed by measuring the relative expression levels of 4 genes encoding glycolysis enzymes. The respiratory chain was studied by assessing the electron transport system and cytochrome c oxidase gene expression. Muscle mitochondria ultrastructure was studied, and antioxidant responses were measured. Furthermore, the main energy reserves-carbohydrates, lipids, and proteins-were measured, and cellular energy was evaluated by measuring ATP, ADP, AMP and IMP concentrations. This study revealed a disturbance of the cell energy metabolism due to copper exposure, with a significant decrease in adenylate energy charge in roach exposed to 10μg/L of copper after 1day. Moreover, ATP concentrations significantly decreased in roach exposed to 10μg/L of copper after 1day. This significant decrease persisted in roach exposed to 50µg/L of copper after 7days. AMP concentrations increased in all contaminated fish after 1day of exposure. In parallel, the relative expression of 3 genes encoding for glycolysis enzymes increased in all contaminated fish after 1day of copper exposure. Focusing on the respiratory chain, cytochrome c oxidase gene expression also increased in all contaminated fish at the two time-points. The activity of the electron transport system was not disturbed by copper, except in roach exposed to 100µg/L of copper after 1day. Copper induced a metabolic stress. Juvenile roach seemed to respond to the ensuing high energy demand by increasing their anaerobic metabolism, but the energy produced by the anaerobic metabolism is unable to compensate for the stress induced by copper after 7days. This multi-marker approach allows us to reach a greater understanding of the effects of copper on the physiological responses of juvenile roach.

Anoxic metabolism and biochemical production in Pseudomonas putida F1 driven by a bioelectrochemical system.

BackgroundPseudomonas putida is a promising host for the bioproduction of chemicals, but its industrial applications are significantly limited by its obligate aerobic character. The aim of this paper is to empower the anoxic metabolism of wild-type Pseudomonas putida to enable bioproduction anaerobically, with the redox power from a bioelectrochemical system (BES).ResultsThe obligate aerobe Pseudomonas putida F1 was able to survive and produce almost exclusively 2–Keto-gluconate from glucose under anoxic conditions due to redox balancing with electron mediators in a BES. 2-Keto-gluconate, a precursor for industrial anti-oxidant production, was produced at an overall carbon yield of over 90 % based on glucose. Seven different mediator compounds were tested, and only those with redox potential above 0.207 V (vs standard hydrogen electrode) showed interaction with the cells. The productivity increased with the increasing redox potential of the mediator, indicating this was a key factor affecting the anoxic production process. P. putida cells survived under anaerobic conditions, and limited biofilm formation could be observed on the anode’s surface. Analysis of the intracellular pools of ATP, ADP and AMP showed that cells had an increased adenylate energy charge suggesting that cells were able to generate energy using the anode as terminal electron acceptor. The analysis of NAD(H) and NADP(H) showed that in the presence of specific extracellular electron acceptors, the NADP(H) pool was more oxidised, while the NAD(H) pool was unchanged. This implies a growth limitation under anaerobic conditions due to a shortage of NADPH and provides a way to limit biomass formation, while allowing cell maintenance and catalysis at high purity and yield.ConclusionsFor the first time, this study proved the principle that a BES-driven bioconversion of glucose can be achieved for a wild-type obligate aerobe. This non-growth bioconversion was in high yields, high purity and also could deliver the necessary metabolic energy for cell maintenance. By combining this approach with metabolic engineering strategies, this could prove to be a powerful new way to produce bio-chemicals and fuels from renewables in both high yield and high purity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0452-y) contains supplementary material, which is available to authorized users.

Characterisation of bioenergetic pathways and related regulators by multiple assays in human tumour cells.

BackgroundAlterations in cellular metabolism are considered as hallmarks of cancers, however, to recognize these alterations and understand their mechanisms appropriate techniques are required. Our hypothesis was to determine whether dominant bioenergetic mechanism may be estimated by comparing the substrate utilisation with different methods to detect the labelled carbon incorporation and their application in tumour cells.MethodsTo define the bioenergetic pathways different metabolic tests were applied: (a) measuring CO2 production from [1-14C]-glucose and [1-14C]-acetate; (b) studying the effect of glucose and acetate on adenylate energy charge; (c) analysing glycolytic and TCA cycle metabolites and the number of incorporated 13C atoms after [U-13C]-glucose/[2-13C]-acetate labelling. Based on [1-14C]-substrate oxidation two selected cell lines out of seven were analysed in details, in which the highest difference was detected at their substrate utilization. To elucidate the relevance of metabolic characterisation the expression of certain regulatory factors, bioenergetic enzymes, mammalian target of rapamycin (mTOR) complexes (C1/C2) and related targets as important elements at the crossroad of cellular signalling network were also investigated.ResultsBoth [U-13C]-glucose and [1-14C]-substrate labelling indicated high glycolytic capacity of tumour cells. However, the ratio of certain 13C-labelled metabolites showed detailed metabolic differences in the two selected cell lines in further characterisation. The detected differences of GAPDH, β-F1-ATP-ase expression and adenylate energy charge in HT-1080 and ZR-75.1 tumour cells also confirmed the altered metabolism. Moreover, the highly limited labelling of citrate by [2-13C]-acetate—representing a novel functional test in malignant cells—confirmed the defect of TCA cycle of HT-1080 in contrast to ZR-75.1 cells. Noteworthy, the impaired TCA cycle in HT-1080 cells were associated with high mTORC1 activity, negligible protein level and activity of mTORC2, high expression of interleukin-1β, interleukin-6 and heme oxygenase-1 which may contribute to the compensatory mechanism of TCA deficiency.ConclusionsThe applied methods of energy substrate utilisation and other measurements represent simple assay system using 13C-acetate and glucose to recognize dominant bioenergetic pathways in tumour cells. These may offer a possibility to characterise metabolic subtypes of human tumours and provide guidelines to find biomarkers for prediction and development of new metabolism related targets in personalized therapy.

Bioenergetic status and oxidative stress during escape response until exhaustion in whiteleg shrimp Litopenaeus vannamei

The escape response in some decapod crustaceans through tail-flipping represents an excellent model of short-term, exhausting muscular activity to understand the role of energy depletion and oxidative stress in muscle fatigue. In this study indicators of oxidative stress and changes in energy metabolism were measured during escape response until fatigue in whiteleg shrimp Litopenaeus vannamei. The escape response of individual shrimp was elicited in a tank by prodding the shrimp with a stick until exhaustion (no further response). A control group consisted of undisturbed shrimp, and a third group was allowed to recover. Arginine phosphate (ArgP) and adenylic energy charge (AEC) were measured by reverse phase HPLC, while fuels, reserves, antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase-GPx), thiobarbituric acid reactive substances, and protein carbonyl content were analyzed by spectrophotometric methods. Shrimp reached fatigue after 31 tail-flips, which decreased abdominal muscle ArgP by 75% and AEC by 18%. Hemolymph lactate levels increased 5 times and muscle GPx activity by 2.7 times. After 1h of recovery, ArgP and AEC base levels were re-established, while hemolymph lactate concentration and GPx activity were still increased, and glucose hemolymph content increased 1.8 times. The drastic decrease in ArgP levels prevents a drop of AEC to a critical level, in accordance with a situation of non-lethal stress. Although no significant changes were observed in muscle glycogen or lactate concentrations, the increase in hemolymph lactate levels indicates an important contribution of muscle glycolysis to prevent a more pronounced decrease in ATP and AEC. The increase in GPx activity in abdominal muscle suggests an increase in reactive oxygen species production during intensive muscular activity, which was adequately neutralized, for no signs of oxidative damage occurred, as indicated by the lack of lipid and protein oxidation. These responses suggest adequate energy efficiency and prevention of oxidative damage during the escape response, in accordance with the adaptative value of such behavior to avoid instantaneous danger, with a capacity of rapid recovery.

Relationship between chronic disturbance of 2,3-diphosphoglycerate metabolism in erythrocytes and Alzheimer disease.

Alzheimer disease (AD) is one of the most common neurodegenerative disorders widely occurring among the elderly. The pathogenic mechanisms involved in the development of this disease are still unknown. In AD, in addition to brain, a number of peripheral tissues and cells are affected, including erythrocytes. In this study, we analyzed glycolytic energy metabolism, antioxidant status, glutathione, adenylate and proteolytic systems in erythrocytes from patients with AD and compared with those from age-matched controls and young adult controls. Glycolytic enzymes hexokinase, phosphofructokinase, bisphosphoglycerate mutase and bisphosphoglycerate phosphatase displayed lower activities in agematched controls, and higher activities in AD patients, as compared to those in young adult control subjects. In both aging and AD, oxidative stress is increased in erythrocytes whereas elevated concentrations of hydrogen peroxide and organic hydroperoxides as well as decreased glutathione/glutathione disulfide ratio and glutathione transferase activity can be detected. These oxidative disturbances are also accompanied by reductions in ATP levels, adenine nucleotide pool size and adenylate energy charge. Caspase-3 and calpain activities in age-matched controls and AD patients were about three times those of young adult controls. 2,3-diphosphoglycerate levels were significantly decreased in AD patients. Taken together these data suggest that AD patients are associated with chronic disturbance of 2,3-diphosphoglycerate metabolism in erythrocytes. These defects may play a central role in pathophysiological processes predisposing elderly subjects to dementia.

The adenylate energy charge as a new and useful indicator of capture stress in chondrichthyans.

Quantifying the physiological stress response of chondrichthyans to capture has assisted the development of fishing practices conducive to their survival. However, currently used indicators of stress show significant interspecific and intraspecific variation in species' physiological responses and tolerances to capture. To improve our understanding of chondrichthyan stress physiology and potentially reduce variation when quantifying the stress response, we investigated the use of the adenylate energy charge (AEC); a measure of available metabolic energy. To determine tissues sensitive to metabolic stress, we extracted samples of the brain, heart, liver, white muscle and blood from gummy sharks (Mustelus antarcticus) immediately following gillnet capture and after 3 h recovery under laboratory conditions. Capture caused significant declines in liver, white muscle and blood AEC, whereas no decline was detected in the heart and brain AEC. Following 3 h of recovery from capture, the AEC of the liver and blood returned to "unstressed" levels (control values) whereas white muscle AEC was not significantly different to that immediately after capture. Our results show that the liver is most sensitive to metabolic stress and white muscle offers a practical method to sample animals non-lethally for determination of the AEC. The AEC is a highly informative indicator of stress and unlike current indicators, it can directly measure the change in available energy and thus the metabolic stress experienced by a given tissue. Cellular metabolism is highly conserved across organisms and, therefore, we think the AEC can also provide a standardised form of measuring capture stress in many chondrichthyan species.

In Situ Analysis of Small Populations of Adherent Mammalian Cells Using Laser Ablation Electrospray Ionization Mass Spectrometry in Transmission Geometry.

Most cultured cells used for biomedical research are cultured adherently, and the requisite detachment prior to biochemical analysis might induce chemical changes. This is especially crucial if accurate metabolic measurements are desired, given the rapid turnover of metabolites in living organisms. There are only a few methods available for the nontargeted in situ analysis of small adherent cell populations. Here we show that laser ablation electrospray ionization (LAESI) mass spectrometry (MS) can be used to analyze adherent cells directly, while still attached to the culture surface. To reduce the size of the analyzed cell population, the spot size constraints of conventional focusing in reflection geometry (rg) LAESI had to be eliminated. By introducing transmission geometry (tg) LAESI and incorporating an objective with a high numerical aperture, spot sizes of 10-20 μm were readily achieved. As few as five adherent cells could be specifically selected for analysis in their culturing environment. The importance of in situ analysis was highlighted by comparing the metabolite composition of adherent versus suspended cells. For example, we observed that cells analyzed adherently yielded higher values for the adenylate energy charge (0.90 ± 0.09 for adherent cells vs 0.09 ± 0.03 for suspended cells). Additionally, due to the smaller focal spot size, tg-LAESI enabled the analysis of ∼20 times smaller cell populations compared to rg-LAESI.

Impact of different temperatures on survival and energy metabolism in the Asian citrus psyllid, Diaphorina citri Kuwayama

Temperature influences the life history and metabolic parameters of insects. Asian citrus psyllid (ACP), Diaphorina citri is a tropical and subtropical pest. ACP invaded new regions around the world and threatened the citrus industry as a vector for Huanglongbing (HLB) disease. ACP is widely distributed and can survive high (up to 45°C) and low temperatures (as low as −6°C). The precise mechanism of temperature tolerance in ACP is poorly understood. We investigated adult survival, cellular energy balance, gene expression, and nucleotide and sugar-nucleotide changes under the effect of different temperature regimes (0°C to 45°C with 5°C intervals). The optimum temperatures for survival were 20 and 25°C. Low temperatures of 0°C and 5°C caused 50% mortality after 2 and 4days respectively, while one day at high temperature (40°C and 45°C) caused more than 95% mortality. The lowest quantity of ATP (3.69±1.6ng/insect) and the maximum ATPase enzyme activities (57.43±7.6μU/insect) were observed at 25°C. Correlation between ATP quantities and ATPase activity was negative. Gene expression of hsp 70, V-type proton ATPase catalytic subunit A and ATP synthase α subunit matched these results. Twenty-four nucleotides and sugar-nucleotides were quantified using HPLC in ACP adults maintained at low, high, and optimum temperatures. The nucleotide profiles were different among treatments. The ratios between AMP:ATP and ADP:ATP were significantly decreased and positively correlated to adults survival, whereas the adenylate energy charge was increased in response to low and high temperatures. Exploring energy metabolic regulation in relation with adult survival might help in understanding the physiological basis of how ACP tolerates newly invaded regions.

Oxygen availability strongly affects chronological lifespan and thermotolerance in batch cultures of Saccharomyces cerevisiae.

Stationary-phase (SP) batch cultures of Saccharomyces cerevisiae, in which growth has been arrested by carbon-source depletion, are widely applied to study chronological lifespan, quiescence and SP-associated robustness. Based on this type of experiments, typically performed under aerobic conditions, several roles of oxygen in aging have been proposed. However, SP in anaerobic yeast cultures has not been investigated in detail. Here, we use the unique capability of S. cerevisiae to grow in the complete absence of oxygen to directly compare SP in aerobic and anaerobic bioreactor cultures. This comparison revealed strong positive effects of oxygen availability on adenylate energy charge, longevity and thermotolerance during SP. A low thermotolerance of anaerobic batch cultures was already evident during the exponential growth phase and, in contrast to the situation in aerobic cultures, was not substantially increased during transition into SP. A combination of physiological and transcriptome analysis showed that the slow post-diauxic growth phase on ethanol, which precedes SP in aerobic, but not in anaerobic cultures, endowed cells with the time and resources needed for inducing longevity and thermotolerance. When combined with literature data on acquisition of longevity and thermotolerance in retentostat cultures, the present study indicates that the fast transition from glucose excess to SP in anaerobic cultures precludes acquisition of longevity and thermotolerance. Moreover, this study demonstrates the importance of a preceding, calorie-restricted conditioning phase in the acquisition of longevity and stress tolerance in SP yeast cultures, irrespective of oxygen availability.

Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.

To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations--both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))--were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.

Energy Charge, Redox State, and Metabolite Turnover in Single Human Hepatocytes Revealed by Capillary Microsampling Mass Spectrometry

Metabolic analysis of single cells to uncover cellular heterogeneity and metabolic noise is limited by the available tools. In this study, we demonstrate the utility of capillary microsampling electrospray ionization mass spectrometry with ion mobility separation for nontargeted analysis of single cells. On the basis of accurate mass measurements and collision cross-section determination, a large number of chemical species, 22 metabolites and 54 lipids, were identified. To assess the cellular response to metabolic modulators, the adenylate energy charge (AEC) levels for control and rotenone treated cells were evaluated. A significant reduction in the AEC values was observed for rotenone treated cells. For the cells under oxidative stress, the mean value for the [reduced glutathione (GSH)]/[oxidized glutathione (GSSG)] ratio was significantly decreased, whereas the distribution of the [uridine diphosphate N-acetylhexosamine (UDP-HexNAc)]/[uridine diphosphate hexose (UDP-hexose)] ratio exhibited dramatic tailing to higher values. Lipid turnover rates were studied by pulse-chase experiments at the single cell level.

Estimation of toxic hazard – A revised Cramer–Ford–Hall decision tree

Aluminium is neurotoxic in humans and has been implicated in several neurological disorders. Chronic use of buffered aspirins, as aspegic, would likely constitute the major human aluminium uptake source. Low-dose aspirin is beneficial in secondary prevention of cardiovascular events, so it is widely used for long periods of time. We studied if oral administration of aspegic to rats modified the aluminium inhibitory effect on brain (Na+/K+)ATPase due to alteration in synaptosomal membrane aluminium content. Adult male Wistar rats were submitted to sub-acute (1.00 g/day during 10 days) and chronic (0.03 g/day during 4 months) dietary AlCl3 exposure and/or to aspegic (0.11 g/day). The exposure protocol increased the synaptosomal aluminium content especially after a long-term exposure to aluminium and aspegic. Although no alterations were observed in rat body weight gain and adenylate energy charge, the (Na+/K+)ATPase activity was significantly reduced when aluminium was orally administered to rats. The oral administration of aspegic increased the synaptosomal aluminium content and concomitantly enhanced the (Na+/K+)ATPase inhibition. In our exposure protocol the increase in synaptosomal aluminium content correlates with the reduction of the (Na+/K+)ATPase activity.